The present invention relates to cloning vectors and methods for producing a library of DNA molecules capable of expressing a fusion polypeptide on the surface of a filamentous phage particle. In particular, the invention relates to display of combinatorial libraries, particularly heterodimeric proteins, using filamentous phage pVII and pIX proteins.
Phage display has been intensively investigated for producing combinatorial antibody libraries and for presentation of combinatorial arrays of peptide elements. See, for example, Rodi et al, Curr. Opin. Biotechnol., 10:87-93, 1999; Vaughan et al, Nat. Biotechnol., 16:535-539, 1998; Griffiths et al, Curr. Opin. Biotechnol., 9:102-108, 1998; Zwick et al, Curr. Opin. Biotechnol., 9:427-436, 1998; Dall""Acqua et al, Curr. Opin. Struct. Biol., 8:443-450, 1998; Raag et al, Faseb J., 9:73-80, 1995; Barbas et al, Proc. Natl. Acad. Sci. USA, 88:7978-7982, 1991; Kang et al, Proc. Natl. Acad. Sci. USA, 88:4363-4366, 1991; Huse et al, Science, 246:1273-1278, 1989).
However, many details of the phage particle itself have not been fully elucidated and the possibility of alternative display formats also remain to be explored. The filamentous bacteriophage fd, and similarly M13, consists of a circular, single-stranded DNA molecule surrounded by a cylinder of coat proteins (FIG. 1). The molecular mass of a particle is about 1.6xc3x97107 Da of which 88% is protein and 12% is DNA (Berkowitz et al, J. Mol. Biol., 102:531-547, 1976). There are about 2700 molecules of the major coat protein pVIII that envelope the phage. At one end of the particle, there are five copies each of pIII and pVI that are involved in host-cell binding and in the termination of the assembly process. The other end contains five copies each of pVII and pIX that are hydrophobic peptides of 33 and 32 amino acids, respectively, required for the initiation of assembly and for maintenance of virion stability. While pIII, pVI, and pVIII have been used to display biological molecules, pVII and pIX have not been utilized (Rodi et al, Curr. Opin. Biotechnol., 10:87-93, 1999; Russel et al, J. Virol., 63:3284-3295, 1989).
Attempts at phage assembly in the absence of pVII and pIX almost completely abolished the production of phage. In addition, prior attempts at displaying a fusion protein on pVII or pIX previously showed that pVII and pIX were not functional with another protein fused to their N-termini (Endemann et al, J. Mol. Biol., 250:496-506, 1995), indicating that display would not be feasible using pVII, pIX, or both.
Despite the enormous attention focused on pIII- and pVIII-mediated phage display, there are no descriptions of the use of pVII or pIX for display of foreign proteins, polypeptides or antigen binding molecules, such as single chain antibodies or components of a heterodimeric protein complex.
It has now been discovered that pVII and pIX can be used for displaying a peptide when fused to the N-termini of either of the two coat proteins. Of greater significance, It described herein that antibody variable regions fused to pVII and pIX engage in a dynamic interaction on the phage surface to display a functional Fv antibody, a representative heterodimeric motif. The display on phage of antibody heavy and light chain variable regions is therefore a prototype for display and assay of diverse libraries of combinatorial heterodimeric arrays in which members can function as dimeric artificial antibody species and allow for selection of novel biological activities.
Artificial antibodies are here defined as protein motifs of large diversity that use the functional strategy of the antibody molecule, but can be free of loop and framework structural constraints. When reduced to its essence, the antibody molecule is a biological device for the presentation of a combinatorial array of peptide elements in three-dimensional space. The essential feature is that while CDRs (complementarity determining regions) cooperate to form a binding site, their interaction is dynamic and functional with little structural association between the CDRs themselves. In this way, the full complement of amino acid residues are available for antigen recognition at a minimum energetic cost for binding. It is proposed that the ability to control the combinatorial design of not only sequence space, but also three-dimensional space, would recapitulate and ultimately transcend the natural design of the immune repertoire.
Thus the invention describes a combinatorial phage display format for construction of highly diverse heterodimeric polypeptide arrays.
In particular, the invention describes a filamentous phage particle encapsulating a genome encoding a fusion polypeptide, wherein the fusion polypeptide comprises an exogenous polypeptide fused to the amino terminus of a filamentous phage pVII or pIX protein. Preferably, the phage particle comprises the expressed fusion protein on the surface of the phage particle.
In a preferred embodiment, the phage genome further encodes a second fusion polypeptide, wherein the second fusion polypeptide comprises a second exogenous polypeptide fused to the amino terminus of the pIX protein and the first exogenous polypeptide in the first fusion polypeptide is fused to the amino terminus of the pVII protein. In this embodiment, the first and second fusion polypeptides can associate to form a heterodimeric protein complex, such as an immunoglobulin Fv, a catalytic Fv, a receptor, a nucleic acid binding protein or an enzyme.
In a related embodiment, the invention describes a vector for expressing a fusion protein on the surface of a filamentous phage comprising a cassette for expressing the fusion protein. The cassette includes upstream and downstream translatable DNA sequences operatively linked via a sequence of nucleotides adapted for directional ligation of an insert DNA, i.e., a polylinker, where the upstream sequence encodes a prokaryotic secretion signal, the downstream sequence encodes a pVII or pIX filamentous phage protein. The translatable DNA sequences are operatively linked to a set of DNA expression signals for expression of the translatable DNA sequences as portions of the fusion polypeptide. In a preferred variation, the vector further comprises a second cassette for expressing a second fusion protein on the surface of the filamentous phage, wherein the second cassette has the structure of the first cassette with the proviso that the first fusion protein expression cassette encodes pVII protein and the second fusion protein expression cassette encodes pIX protein. The vector is used as a phage genome to express heterodimeric protein complexes on the surface of the phage particle in which the two exogenous polypeptides of the heterodimer are anchored on the phage particle by the fusion to the first and second phage proteins, pVII and pIX, respectively.
In another embodiment, the invention contemplates a library of phage particles according to the present invention, i.e., a combinatorial library, in which representative particles in the library each display a different fusion protein. Where the particle displays a heterodimeric protein complex, the library comprises a combinatorial library of heterodimers, such as antibodies in the form of a library of Fv molecules. Preferred libraries have a diversity of at least 107 different species of fusion protein.
A related embodiment describes a fusion protein comprising first and second polypeptides wherein the first polypeptide is an exogenous protein and the second polypeptide is a filamentous phage pVII or pIX protein, wherein the exogenous protein is fused to the amino terminus of the filamentous phage protein.
Still further, the invention contemplates a variety of methods for producing a combinatorial library of phage, including by cloning repertoires of genes encoding an exogenous polypeptide into a vector of the present invention, modifying the structure of the exogenous polypeptides in a library by mutagenesis, by random combination of populations of first and second fusion protein libraries, by affinity selection (xe2x80x9cpanningxe2x80x9d) to alter the diversity of a library, and the like.
The design of proteins with improved or novel functions is an important goal with a variety of medical, industrial, environmental, and basic research applications. Following the development of combinatorial antibody libraries, a powerful next step is the evolution toward artificial antibody constructs as well as other protein motifs in which dimeric species are native or might be functional.
The present invention addresses these challenges by providing a phage-display format for the construction of combinatorial heterodimeric polypeptide arrays in which pVII and pIX are utilized for the display of fusion proteins that form dimeric species. It is important to note that this is an entirely new methodology because one can independently display two protein motifs in close proximity to generate a library of functional interactions.
Inherent in the scope and power of the technology is the ability to display a variety of proteins that can engage in dimeric interactions. These include not only antibodies, but also some enzymes, hormones and hormone receptors, and DNA-binding proteins. The display technology described herein can be used for combinatorial alteration of antibody framework regions and to reorganize and miniaturize the antibody structure or to display DNA binding proteins, such as repressors, as a library of heterodimers for selection against particular DNA sequences of clinical and therapeutic importance.
Thus the present technology provides for the display and selection of mutant dimeric proteins and combinatorial libraries in which members consist of heterodimeric arrays. Using this technology, the native immunoglobulin structure, in a heterodimeric VH-VL Fv format shown herein, can be modified in different ways and screened for specificity and activity. For example, by combinatorial alteration of framework regions (FRs) or other manipulations to reorganize and miniaturize the antibody structure by processes coined xe2x80x9ccomplementarity determining region (CDR) shufflingxe2x80x9d and xe2x80x9ctwinibodyxe2x80x9d formation, antibody-like secondary structures will emerge that contain new paratopes or entirely different structural elements. Selection for binding and/or catalysis against the natural antigen and/or substrate as well as some related compounds will be used to screen the libraries of heterodimeric proteins.
Furthermore, sequence randomizations to form libraries and chain-shuffling protocols to form hybrid species can lead to subsets of novel proteins. For instance, the display and modification of arrays of zinc-finger domains in homodimeric or heterodimeric form produces structures which possess specific DNA interactions. In addition, entirely new constructs are possible via the insertion of a desired encoding fragment within a preformed scaffold such as an antibody chain. Possible insertions include an enzyme signature sequence or a repressor binding protein.
It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.